Download Bio II Ch 20 DNA Technology

CHAPTER 20 DNA TECHNOLOGY
AND GENOMICS
Section A: DNA Cloning
1. DNA technology makes it possible to clone genes for basic research and
commercial applications: an overview
2. Restriction enzymes are used to make recombinant DNA
3. Genes can be clones in recombinant DNA vectors: a closer look
4. Cloned genes are stored in DNA libraries
5. The polymerase chain reaction (PCR) closed DNA directly in vitro
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Introduction
• The mapping and sequencing of the human genome has
been made possible by advances in DNA technology.
• Progress began with the development of techniques for
making recombinant DNA, in which genes from two
different sources - often different species - are combined
in vitro into the same molecule.
• These methods form part of genetic engineering, the
direct manipulation of genes for practical purposes.
• Applications include the introduction of a desired gene
into the DNA of a host that will produce the desired
protein.
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• DNA technology has launched a revolution in
biotechnology, the manipulation of organisms or
their components to make useful products.
• Practices that go back centuries, such as the use of
microbes to make wine and cheese and the selective
breeding of livestock, are examples of biotechnology.
• Biotechnology based on the manipulation of DNA in
vitro differs from earlier practices by enabling scientists
to modify specific genes and move them between
organisms as distinct as bacteria, plants, and animals.
• DNA technology is now applied in areas ranging
from agriculture to criminal law, but its most
important achievements are in basic research.
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• To study a particular gene, scientists needed to
develop methods to isolate only the small, welldefined, portion of a chromosome containing the
gene.
• Techniques for gene cloning enable scientists to
prepare multiple identical copies of gene-sized
pieces of DNA.
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• 1. DNA technology makes it possible to
clone genes for basic research and
commercial applications: an overview
• Most methods for cloning pieces of DNA share
certain general features.
• For example, a foreign gene is inserted into a bacterial
plasmid and this recombinant DNA molecule is
returned to a bacterial cell.
• Every time this cell reproduces, the recombinant plasmid is
replicated as well and passed on to its descendents.
• Under suitable conditions, the bacterial clone will make the
protein encoded by the foreign gene.
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• One basic cloning technique begins with the
insertion of a foreign gene into a bacterial plasmid.
Fig. 20.1
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• The potential uses of cloned genes fall into two
general categories.
• First, the goal may be to produce a protein product.
• For example, bacteria carrying the gene for human
growth hormone can produce large quantities of the
hormone for treating stunted growth.
• Alternatively, the goal may be to prepare many
copies of the gene itself.
• This may enable scientists to determine the gene’s
nucleotide sequence or provide an organism with a new
metabolic capability by transferring a gene from another
organism.
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2. Restriction enzymes are used to make
recombinant DNA
• Gene cloning and genetic engineering were made
possible by the discovery of restriction enzymes
that cut DNA molecules at specific locations.
• In nature, bacteria use restriction enzymes to cut
foreign DNA, such as from phages or other bacteria.
• Most restrictions enzymes are very specific,
recognizing short DNA nucleotide sequences and
cutting at specific point in these sequences.
• Bacteria protect their own DNA by methylation.
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• The enzymatic function of restriction enzymes
is to cleave nucleic acids at specific sites.
• These are often a symmetrical series of four to eight
bases on both strands running in opposite directions.
• If the restriction site on one strand is 3’-CTTAAG-5’,
the complementary strand is 5’-GAATTC-3’.
• Because the target sequence usually occurs (by
chance) many times on a long DNA molecule, an
enzyme will make many cuts.
• Copies of a DNA molecule will always yield the same
set of restriction fragments when exposed to a specific
enzyme.
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• Restriction enzymes cut covalent phosphodiester
bonds of both strands, often in a staggered way
creating single-stranded ends, sticky ends.
• These extensions will form hydrogen-bonded base pairs
with complementary single-stranded stretches on other
DNA molecules cut with the same restriction enzyme.
• These DNA fusions can be made permanent by
DNA ligase which seals the strand by catalyzing
the formation of phosphodiester bonds.
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• Restriction enzymes
and DNA ligase can
be used to make
recombinant DNA,
DNA that has been
spliced together from
two different sources.
Fig. 20.2
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3. Genes can be cloned in recombinant
DNA vectors: a closer look
• Recombinant plasmids are produced by splicing
restriction fragments from foreign DNA into
plasmids.
• These can be returned relatively easily to bacteria.
• The original plasmid used to produce recombinant DNA is
called a cloning vector, which is a DNA molecule that
can carry foreign DNA into a cell and replicate there.
• Then, as a bacterium carrying a recombinant plasmid
reproduces, the plasmid replicates within it.
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• Bacteria are most commonly used as host cells for
gene cloning because DNA can be easily isolated
and reintroduced into their cells.
•Bacteria containing recombinant plasmids
are often identified by exposing the bacteria to
an antibiotic that kills cells lacking the
plasmid.
• Bacteria cultures also grow quickly, rapidly
replicating the foreign genes.
• The process of
cloning a human
gene in a bacterial
plasmid can be
divided into five
steps.
Fig. 20.3
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1. Isolation of vector and gene-source DNA.
• The source DNA comes from human tissue cells.
• The source of the plasmid is typically E. coli.
• This plasmid carries two useful genes, ampR, conferring
resistance to the antibiotic ampicillin and lacZ, encoding the
enzyme beta-galactosidase which catalyzes the hydrolysis of
sugar.
• The plasmid has a single recognition sequence, within the
lacZ gene, for the restriction enzyme used.
• Plasmids are important in biotechnology because they
are a vehicle for the insertion of foreign genes into
bacteria. If you discovered a bacterial cell that contained
no restriction enzymes, the cell would be easily infected
and lysed by bacteriophages.
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2. Insertion of DNA into the vector.
• By digesting both the plasmid and human DNA
with the same restriction enzyme we can create
thousands of human DNA fragments, one fragment
with the gene that we want, and with compatible
sticky ends on bacterial plasmids.
• After mixing, the human fragments and cut
plasmids form complementary pairs that are then
joined by DNA ligase.
• This creates a mixture of recombinant DNA
molecules.
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3. Introduction of the cloning vector into cells.
• Bacterial cells take up the recombinant plasmids
by transformation.
• These bacteria are lacZ-, unable to hydrolyze lactose.
• This creates a diverse pool of bacteria, some
bacteria that have taken up the desired recombinant
plasmid DNA, other bacteria that have taken up
other DNA, both recombinant and
nonrecombinant.
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4. Cloning of cells (and foreign genes).
• We can plate out the transformed bacteria on a
solid nutrient medium containing ampicillin and a
sugar called X-gal.
• Only bacteria that have the ampicillin-resistance
plasmid will grow.
• The X-gal in the medium is used to identify plasmids
that carry foreign DNA.
• Bacteria with plasmids lacking foreign DNA stain
blue when beta-galactosidase hydrolyzes X-gal.
• Bacteria with plasmids containing foreign DNA are
white because they lack beta-galactosidase.
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5. Identifying cell clones with the right gene.
• In the final step, we will sort through the thousands
of bacterial colonies with foreign DNA to find
those containing our gene of interest.
• One technique, nucleic acid hybridization,
depends on base pairing between our gene and a
complementary sequence, a nucleic acid probe,
on another nucleic acid molecule.
• The sequence of our RNA or DNA probe depends on
knowledge of at least part of the sequence of our gene.
• A radioactive or fluorescent tag labels the probe.
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• The probe will
hydrogen-bond
specifically to
complementary
single strands of
the desired gene.
• After denaturation
(separating) the DNA
strands in the plasmid,
the probe will bind
with its complementary
sequence, tagging
colonies with the
targeted gene.
Fig. 20.4
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• Because of different details between prokaryotes
and eukaryotes, inducing a cloned eukaryotic gene
to function in a prokaryotic host can be difficult.
• One way around this is to employ an expression
vector, a cloning vector containing the requisite
prokaryotic promotor upstream of the restriction site.
• The bacterial host will then recognize the promotor and
proceed to express the foreign gene that has been linked
to it, including many eukaryotic proteins.
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• The presence of introns, long non-coding regions,
in eukaryotic genes creates problems for
expressing these genes in bacteria.
• To express eukaryotic genes in bacteria, a fully
processed mRNA acts as the template for the synthesis
of a complementary strand using reverse transcriptase.
• This complementary DNA (cDNA), with a promoter,
can be attached to a vector for replication, transcription,
and translation inside bacteria.
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• Complementary
DNA is DNA
made in vitro
using mRNA as a
template and the
enzyme reverse
transcriptase.
Fig. 20.5
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• Molecular biologists can avoid incompatibility
problems by using eukaryotic cells as host for
cloning and expressing eukaryotic genes.
• Yeast cells, single-celled fungi, are as easy to grow
as bacteria and have plasmids, rare for eukaryotes.
• Scientists have constructed yeast artificial
chromosomes (YACs) - an origin site for
replication, a centromere, and two telomeres with foreign DNA.
• These chromosomes behave normally in mitosis
and can carry more DNA than a plasmid.
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• Another advantage of eukaryotic hosts is that they
are capable of providing the posttranslational
modifications that many proteins require.
• This includes adding carbohydrates or lipids.
• For some mammalian proteins, the host must be an
animal or plant cell to perform the necessary
modifications.
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• Many eukaryotic cells can take up DNA from their
surroundings, but often not efficiently.
• Several techniques facilitate entry of foreign DNA.
• In electroporation, brief electrical pulses create a
temporary hole in the plasma membrane through which
DNA can enter.
• Alternatively, scientists can inject DNA into individual
cells using microscopically thin needles.
• In a technique used primarily for plants, DNA is attached
to microscopic metal particles and fired into cells with a
gun.
• Once inside the cell, the DNA is incorporated into the
cell’s DNA by natural genetic recombination.
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4. Cloned genes are stored in DNA
libraries
• In the “shotgun” cloning approach, a mixture of
fragments from the entire genome is included in
thousands of different recombinant plasmids.
• A complete set of recombinant plasmid clones, each
carrying copies of a particular segment from the
initial genome, forms a genomic library.
• The library can be saved and used as a source of other
genes or for gene mapping.
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• In addition to plasmids, certain bacteriophages are
also common cloning vectors for making libraries.
• Fragments of foreign DNA can be spliced into a phage
genome using a restriction enzyme and DNA ligase.
• The recombinant phage
DNA is packaged in a
capsid in vitro and
allowed to infect a
bacterial cell.
• Infected bacteria
produce new phage
particles, each with
the foreign DNA.
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• A more limited kind of gene library can be
developed from complementary DNA.
• During the process of producing cDNA, all mRNAs are
converted to cDNA strands.
• This cDNA library represents that part of a cell’s
genome that was transcribed in the starting cells.
• The polymerase chain reaction (PCR) has been used
to amplify DNA from any organism: fossils, fetal
cells, viruses, or bacteria.
• By making cDNA libraries from cells of the same type
at different times in the life of an organism, one can
trace changes in the patterns of gene expression.
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5. The polymerase chain reaction (PCR)
clones DNA entirely in vitro
• DNA cloning is the best method for preparing large
quantities of a particular gene or other DNA
sequence.
• When the source of DNA is scanty or impure, the
polymerase chain reaction (PCR) is quicker and
more selective.
• The polymerase chain reaction is important
because it allows us to make many copies of a
targeted segment of DNA. This technique can
quickly amplify any piece of DNA without using
cells.
• The DNA is
incubated in a
test tube with
special DNA
polymerase, a
supply of
nucleotides,
and short
pieces of
single-stranded
DNA as
a primer.
Fig. 20.7
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• PCR can make billions of copies of a targeted
DNA segment in a few hours.
• This is faster than cloning via recombinant bacteria.
• In PCR, a three-step cycle--heating, cooling, and
replication--brings about a chain reaction that
produces an exponentially growing population of
DNA molecules.
• The key to easy PCR automation was the discovery of
an unusual DNA polymerase, isolated from bacteria
living in hot springs, which can withstand the heat
needed to separate the DNA strands at the start of each
cycle.
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• PCR is very specific.
• By their complementarity to sequences bracketing
the targeted sequence, the primers determine the
DNA sequence that is amplified.
• PCR can make many copies of a specific gene before
cloning in cells, simplifying the task of finding a clone
with that gene.
• PCR is so specific and powerful that only minute
amounts of DNA need be present in the starting material.
• Occasional errors during PCR replication impose
limits to the number of good copies that can be
made when large amounts of a gene are needed.
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• Devised in 1985, PCR has had a major impact on
biological research and technology.
• PCR has amplified DNA from a variety of sources:
• Fragments of ancient DNA from a 40,000-year-old
frozen woolly mammoth.
• DNA from tiny amount of blood or semen found at the
scenes of violent crimes.
• DNA from single embryonic cells for rapid prenatal
diagnosis of genetic disorders.
• DNA of viral genes from cells infected with difficult-todetect viruses such as HIV.
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PCR Summary
• The complete sequence of steps occurring
during every cycle of PCR is:
1) The mixture is heated to a high temperature
to denature the double stranded target DNA
2) The primers hybridize to the target DNA
3) DNA polymerase extends the primers to make
a copy of the target DNA.
You never have to add fresh DNA polymerase
because it is used repeatedly.
CHAPTER 20 DNA TECHNOLOGY
AND GENOMICS
Section B: DNA Analysis and Genomics
1. Restriction fragment analysis detects DNA differences that affect restriction
sites
2. Entire genomes can be mapped at the DNA level
3. Genomic sequences provide clues to important biological questions
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Introduction
• Once we have prepared homogeneous samples of
DNA, each containing a large number of identical
segments, we can begin to ask some far-ranging
questions.
• These include:
• Are there differences in a gene in different people?
• Where and when is a gene expressed?
• What is the the location of a gene in the genome?
• How has a gene evolved as revealed in interspecific
comparisons?
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• To answer these questions, we will eventually need
to know the nucleotide sequence of the gene and
ultimately the sequences of entire genomes.
• Comparisons among whole sets of genes and their
interactions is the field of genomics.
• One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis.
• Gel electrophoresis separates macromolecules - nucleic
acids or proteins - on the basis of their rate of movement
through a gel in an electrical field.
• Rate of movement depends on size, electrical charge, and
other physical properties of the macromolecules.
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• For linear DNA molecules, separation depends
mainly on size (length of fragment) with longer
fragments migrating less along the gel.
Fig. 20.8
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1. Restriction fragment analysis detects DNA
differences that affect restriction sites
• Restriction fragment analysis indirectly detects
certain differences in DNA nucleotide sequences.
• After treating long DNA molecules with a restriction
enzyme, the fragments can be separated by size via gel
electrophoresis.
• This produces a series of bands that are characteristic of
the starting molecule and that restriction enzyme.
• The separated fragments can be recovered undamaged
from gels, providing pure samples of individual
fragments.
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• We can use restriction fragment analysis to
compare two different DNA molecules
representing, for example, different alleles.
• Because the two alleles must differ slightly in DNA
sequence, they may differ in one or more restriction
sites.
• If they do differ in restriction sites, each will produce
different-sized fragments when digested by the same
restriction enzyme.
• In gel electrophoresis, the restriction fragments from the
two alleles will produce different band patterns,
allowing us to distinguish the two alleles.
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• Restriction fragment analysis is sensitive enough to
distinguish between two alleles of a gene that differ by
only base pair in a restriction site.
Fig. 20.9
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• Gel electrophoresis combined with nucleic acid
hybridization allows analyses to be conducted on
the whole genome, not just cloned and purified
genes.
• Although electrophoresis will yield too many
bands to distinguish individually, we can use
nucleic acid hybridization with a specific probe to
label discrete bands that derive from our gene of
interest.
• The radioactive label on the single-stranded probe
can be detected by autoradiography, identifying the
fragments that we are interested in.
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• We can tie together several molecular techniques
to compare DNA samples from three individuals.
• We start by adding the restriction enzyme to each of the
three samples to produce restriction fragments.
• We then separate the fragments by gel electrophoresis.
• Southern blotting (Southern hybridization) allows us
to transfer the DNA fragments from the gel to a sheet of
nitrocellulose paper, still separated by size.
• This also denatures the DNA fragments.
• Bathing this sheet in a solution containing our probe
allows the probe to attach by base-pairing (hybridize) to
the DNA sequence of interest and we can visualize
bands containing the label with autoradiography.
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• For our three individuals, the results of these steps show
that individual III has a different restriction pattern than
individuals I or II.
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Fig. 20.10
• Southern blotting can be used to examine
differences in noncoding DNA as well.
• Differences in DNA sequence on homologous
chromosomes that produce different restriction
fragment patterns are scattered abundantly
throughout genomes, including the human genome.
• These restriction fragment length polymorphisms
(RFLPs) can serve as a genetic marker for a
particular location (locus) in the genome.
• A given RFLP marker frequently occurs in numerous
variants in a population.
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• RFLPs are detected and analyzed by Southern
blotting, frequently using the entire genome as the
DNA starting material.
• These techniques will detect RFLPs in noncoding or
coding DNA.
• Because RFLP markers are inherited in a
Mendelian fashion, they can serve as genetic
markers for making linkage maps.
• The frequency with which two RFPL markers - or a
RFLP marker and a certain allele for a gene - are
inherited together is a measure of the closeness of the
two loci on a chromosome.
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2. Entire genomes can be mapped at the
DNA level
• As early as 1980, Daniel Botstein and colleagues
proposed that the DNA variations reflected in RFLPs
could serve as the basis of an extremely detailed map
of the entire human genome.
• For some organisms, researchers have succeeded in
bringing genome maps to the ultimate level of detail:
the entire sequence of nucleotides in the DNA.
• They have taken advantage of all the tools and techniques
already discussed - restriction enzymes, DNA cloning, gel
electrophoresis, labeled probes, and so forth.
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• One ambitious research project made possible by
DNA technology has been the Human Genome
Project, begun in 1990.
• This is an effort to map the entire human genome,
ultimately by determining the complete nucleotide
sequence of each human chromosome.
• An international, publicly funded consortium has
proceeded in three phases: genetic (linkage) mapping,
physical mapping, and DNA sequencing.
• In addition to mapping human DNA, the genomes
of other organisms important to biological research
are also being mapped.
• These include E. coli, yeast, fruit fly, and mouse.
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• In mapping a large genome, the first stage is to
construct a linkage map of several thousand
markers spaced throughout the chromosomes.
• The order of the markers and the relative distances
between them on such a map are based on
recombination frequencies.
• The markers can be genes or any other identifiable
sequences in DNA, such as RFLPs or microsatellites.
• The human map with 5,000 genetic markers
enabled researchers to locate other markers,
including genes, by testing for genetic linkage with
the known markers.
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• The next step was converting the relative distances
to some physical measure, usually the number of
nucleotides along the DNA.
• For whole-genome mapping, a physical map is
made by cutting the DNA of each chromosome
into identifiable restriction fragments and then
determining the original order of the fragments.
• The key is to make fragments that overlap and then use
probes or automated nucleotide sequencing of the ends
to find the overlaps.
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• In chromosome
walking, the
researcher starts
with a known DNA
segment (cloned,
mapped, and
sequenced) and
“walks” along the
DNA from that
locus, producing a
map of overlapping
fragments.
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Fig. 20.11
• When working with large genomes, researchers
carry out several rounds of DNA cutting, cloning,
and physical mapping.
• The first cloning vector is often a yeast artificial
chromosome (YAC), which can carry inserted
fragments up to a million base pairs long, or a bacterial
artificial chromosome (BAC), which can carry inserts
of 100,000 to 500,000 base pairs.
• After the order of these long fragments has been
determined (perhaps by chromosome walking), each
fragment is cut into pieces, which are cloned and
ordered in turn.
• The final sets of fragments, about 1,000 base pairs long,
are cloned in plasmids or phage and then sequenced.
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• The complete nucleotide sequence of a genome is
the ultimate map.
• Starting with a pure preparation of many copies of a
relatively short DNA fragment, the nucleotide sequence
of the fragment can be determined by a sequencing
machine.
• The usual sequencing technique combines DNA
labeling, DNA synthesis with special chain-terminating
nucleotides, and high resolution gel electrophoresis.
• A major thrust of the Human Genome Project has been
the development of technology for faster sequencing
and more sophisticated software for analyzing and
assembling the partial sequences.
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• One common method of sequencing DNA, the
Sanger method, is similar to PCR.
• However, inclusion of special dideoxynucleotides
in the reaction mix ensures that rather than copying
the whole template, fragments of various lengths
will be synthesized.
• These dideoxynucleotides, marked radioactively or
fluorescently, terminate elongation when they are
incorporated randomly into the growing strand because
they lack a 3’-OH to attach the next nucleotide.
• The order of these fragments via gel
electrophoresis can be interpreted as the nucleotide
sequence.
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Fig. 20.12
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• While the public consortium has followed a
hierarchical, three-stage approach for sequencing
an entire genome, J. Craig Venter decided in 1992
to try a whole-genome shotgun approach.
• This uses powerful computers to assemble sequences
from random fragments, skipping the first two steps.
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Fig. 20.13
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• The worth of his approach was demonstrated in
1995 when he and colleagues reported the
complete sequence of a bacterium.
• His private company, Celera Genomics, finished
the sequence of Drosophila melanogaster in 2000.
• In February, 2001, Celera and the public
consortium separately announced sequencing over
90% of the human genome.
• Competition and an exchange of information and
approaches between the two groups has hastened
progress.
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• By mid-2001, the genomes of about 50 species had
been completely (or almost completely) sequenced.
• They include E. coli and a number of other bacteria and
several archaea.
• Sequenced eukaryotes include a yeast, a nematode, and a
plant Arabidopsis thaliana.
• There are still many gaps in the human sequence.
• Areas with repetitive DNA and certain parts of the
chromosomes of multicellular organisms resist detailed
mapping by the usual methods.
• On the other hand, the sequencing of the mouse genome
(about 85% identical to the human genome) is being
greatly aided by knowledge of the human sequence.
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3. Genome sequences provide clues to
important biological questions
• Genomics, the study of genomes based on their DNA
sequences, is yielding new insights into fundamental
questions about genome organization, the control of
gene expression, growth and development, and
evolution.
• Rather than inferring genotype from phenotype like
classical geneticists, molecular geneticists try to
determine the impact on the phenotype of details of
the genotype.
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• DNA sequences, long lists of A’s, T’s, G’s,and
C’s, are being collected in computer data banks
that are available to researchers everywhere via the
Internet.
• Special software can scan the sequences for the
telltale signs of protein-coding genes, such as start
and stop signals for transcription and translation,
and those for RNA-splicing sites.
• From these expressed sequence tags (ESTs),
researchers can collect a list of gene candidates.
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• The surprising -- and humbling -- result to date
from the Human Genome Project is the small
number of putative genes, 30,000 to 40,000.
• This is far less than expected and only two to three
times the number of
genes in the fruit fly
or nematodes.
• Humans have
enormous amounts
of noncoding DNA,
including repetitive
DNA and unusually
long introns.
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• By doing more mixing and matching of modular
elements, humans -- and vertebrates in general -reach more complexity than flies or worms.
• The typical human gene probably specifies at least two
or three different polypeptides by using different
combinations of exons.
• Along with this is additional polypeptide diversity via
post-translational processing.
• The human sequence suggests that our polypeptides tend
to be more complicated than those of invertebrates.
• While humans do not seem to have more types of
domains, the domains are put together in many more
combinations.
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• About half of the human genes were already known
before the Human Genome Project.
• To determine what the others are and what they
may do, scientists compare the sequences of new
gene candidates with those of known genes.
• In some cases, the sequence of a new gene candidate will
be similar in part with that of known gene, suggesting
similar function.
• In other cases, the new sequences will be similar to a
sequence encountered before, but of unknown function.
• In still other cases, the sequence is entirely unlike
anything ever seen before.
• About 30% of the E. coli genes are new to us.
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• Scientists often deduce the evolutionary history
of the different members of a gene family by
comparing the sequences of the genes.
• For example, yeast has a number of genes close enough
to the human versions that they can substitute for them
in a human cell.
• Researchers may determine what a human disease gene
does by studying its normal counterpart in yeast.
• Bacterial sequences reveal unsuspected metabolic
pathways that may have industrial or medical uses.
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• Studies of genomes have also revealed how genes
act together to produce a functioning organism
through an unusually complex network of
interactions among genes and their products.
• To determine which genes are transcribed under
different situations, researchers isolate mRNA from
particular cells and use the mRNA as templates to
build a cDNA library.
• This cDNA can be compared to other collections of
DNA by hybridization.
• This will reveal which genes are active at different
developmental stages, in different tissues, or in tissues in
different states of health.
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• Automation has allowed scientists to detect and
measure the expression of thousands of genes at
one time using DNA microarray assays.
• Tiny amounts of a large number of single-stranded
DNA fragments representing different genes are fixed
on a glass slide in a tightly spaced array (grid).
• The fragments are tested for hybridization with various
samples of fluorescently labeled cDNA molecules.
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Fig. 20.14a
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• Spots where any of the cDNA hybridizes fluoresce
with an intensity indicating the relative amount of
the mRNA that was in the tissue.
Fig. 20.14b
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• Ultimately, information from microarray assays
should provide us a grander view: how ensembles
of genes interact to form a living organism.
• It already has confirmed the relationship between
expression of genes for photosynthetic enzymes and
tissue function in leaves versus roots of the plant
Arabidopsis.
• In other cases, DNA microarray assays are being used
to compare cancerous versus noncancerous tissues.
• This may lead to new diagnostic techniques and
biochemically targeted treatments, as well as a fuller
understanding of cancer.
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• Perhaps the most interesting genes discovered in
genome sequencing and expression studies are
those whose function is completely mysterious.
• One way to determine their function is to disable
the gene and hope that the consequences provide
clues to the gene’s normal function.
• Using in vitro mutagenesis, specific changes are
introduced into a cloned gene, altering or destroying its
function.
• When the mutated gene is returned to the cell, it may be
possible to determine the function of the normal gene
by examining the phenotype of the mutant.
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• In nonmammalian organisms, a simpler and faster
method, RNA interference (RNAi), has been
applied to silence the expression of selected genes.
• This method uses synthetic double-stranded RNA
molecules matching the sequences of a particular gene
to trigger breakdown of the gene’s mRNA.
• The mechanism underlying RNAi is still unknown.
• Scientists have only recently achieved some success in
using the method to silence genes in mammalian cells.
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• The next step after mapping and sequencing
genomes is proteomics, the systematic study of full
protein sets (proteomes) encoded by genomes.
• One challenge is the sheer number of proteins in humans
and our close relatives because of alternative RNA
splicing and post-translational modifications.
• Collecting all the proteins will be difficult because a
cell’s proteins differ with cell type and its state.
• In addition, unlike DNA, proteins are extremely varied in
structure and chemical and physical properties.
• Because proteins are the molecules that actually carry out
cell activities, we must study them to learn how cells and
organisms function.
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• Genomic and proteomics are giving biologists an
increasingly global perspective on the study of life.
• Eric Lander and Robert Weinberg predict that
complete catalogs of genes and proteins will change
the discipline of biology dramatically.
• “For the first time in a century, reductionists [are
yielding] ground to those trying to gain a holistic view of
cells and tissues.”
• Advances in bioinformatics, the application of
computer science and mathematics to genetic and
other biological information, will play a crucial role
in dealing with the enormous mass of data.
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• These analyses will provide understanding of the
spectrum of genetic variation in humans.
• Because we are all probably descended from a small
population living in Africa 150,000 to 200,000 years ago,
the amount of DNA variation in humans is small.
• Most of our diversity is in the form of single nucleotide
polymorphisms (SNPs), single base-pair variations.
• In humans, SNPs occur about once in 1,000 bases,
meaning that any two humans are 99.9% identical.
• The locations of the human SNP sites will provide useful
markers for studying human evolution and for
identifying disease genes and genes that influence our
susceptibility to diseases, toxins or drugs.
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CHAPTER 20 DNA TECHNOLOGY
AND GENOMICS
Section C: Practical Applications of DNA Technology
1. DNA technology is reshaping medicine and the pharmaceutical industry
2. DNA technology offers forensic, environmental, and agricultural
applications
3. DNA technology raises important safety and ethical questions
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1. DNA technology is reshaping medicine
and the pharmaceutical industry
• Modern biotechnology is making enormous
contributions to both the diagnosis of diseases and in
the development of pharmaceutical products.
• The identification of genes whose mutations are
responsible for genetic diseases could lead to ways to
diagnose, treat, or even prevent these conditions.
• Susceptibility to many “nongenetic” diseases, from
arthritis to AIDS, is influenced by a person’s genes.
• Diseases of all sorts involve changes in gene expression.
• DNA technology can identify these changes and lead to
the development of targets for prevention or therapy.
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• PCR and labeled probes can track down the
pathogens responsible for infectious diseases.
• For example, PCR can amplify and thus detect HIV
DNA in blood and tissue samples, detecting an
otherwise elusive infection.
• Medical scientists can use DNA technology to
identify individuals with genetic diseases before
the onset of symptoms, even before birth.
• It is also possible to identify symptomless carriers.
• Genes have been cloned for many human diseases,
including hemophilia, cystic fibrosis, and Duchenne
muscular dystrophy.
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• Hybridization analysis makes it possible to detect
abnormal allelic forms of genes, even in cases in
which the gene has not yet been cloned.
• The presence of an abnormal allele can be diagnosed
with reasonable accuracy if a closely linked RFLP
marker has been found.
• The closeness of the marker to the gene makes crossing
over between them unlikely and the
marker and gene
will almost
always stay
together in
inheritance.
Fig. 20.15
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• Techniques for gene manipulation hold great
potential for treating disease by gene therapy.
• This alters an afflicted individual’s genes.
• A normal allele is inserted into somatic cells of a tissue
affected by a genetic disorder.
• For gene therapy of somatic cells to be permanent, the
cells that receive the normal allele must be ones that
multiply throughout the patient’s life.
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• Bone marrow cells, which include the stem cells
that give rise to blood and immune system cells,
are prime candidates for gene therapy.
• A normal allele could be
inserted by a viral vector
into some bone marrow
cells removed from the
patient.
• If the procedure succeeds,
the returned modified cells
will multiply throughout
the patient’s life and
express the normal gene,
providing missing proteins.
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Fig. 20.16
• Despite “hype” in the news media over the past
decade, there has been very little scientifically
strong evidence of effective gene therapy.
• Even when genes are successfully and safely transferred
and expressed in their new host, their activity typically
diminishes after a short period.
• Most current gene therapy trials are directed not at
correcting genetic defects, but to fight major killers
such as heart disease and cancer.
• The most promising trials are those in which a limited
activity period is not only sufficient but desirable.
• Some success has been reported in stimulated new heart
blood vessels in pigs after gene therapy.
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• Gene therapy poses many technical questions.
• These include regulation of the activity of the transferred
gene to produce the appropriate amount of the gene
product at the right time and place.
• In addition, the insertion of the therapeutic gene must not
harm some other necessary cell function.
• Gene therapy raises some difficult ethical and social
questions.
• Some critics suggest that tampering with human genes,
even for those with life-threatening diseases, is wrong.
• They argue that this will lead to the practice of eugenics,
a deliberate effort to control the genetic makeup of
human populations.
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• The most difficult ethical question is whether we
should treat human germ-line cells to correct the
defect in future generations.
• In laboratory mice, transferring foreign genes into egg
cells is now a routine procedure.
• Once technical problems relating to similar genetic
engineering in humans are solved, we will have to face
the question of whether it is advisable, under any
circumstances, to alter the genomes of human germ
lines or embryos.
• Should we interfere with evolution in this way?
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• From a biological perspective, the elimination of
unwanted alleles from the gene pool could
backfire.
• Genetic variation is a necessary ingredient for the
survival of a species as environmental conditions
change with time.
• Genes that are damaging under some conditions could
be advantageous under other conditions, for example
the sickle-cell allele.
• The principal problem with inserting an unmodified
mammalian gene into a bacterial plasmid, and then
getting that gene expressed in bacteria, is that
bacteria cannot remove eukaryotic introns.
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• DNA technology has been used to create many
useful pharmaceuticals, mostly proteins.
• By transferring the gene for a protein into a host
that is easily grown in culture, one can produce
large quantities of normally rare proteins.
• By including highly active promotors (and other control
elements) into vector DNA, the host cell can be induced
to make large amounts of the product of a gene into the
vector.
• In addition, host cells can be engineered to secrete a
protein, simplifying the task of purification.
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• One of the first practical applications of gene
splicing was the production of mammalian
hormones and other mammalian regulatory proteins
in bacteria.
• These include human insulin and growth factor (HFG).
• Human insulin, produced by bacteria, is superior for the
control of diabetes than the older treatment of pig or
cattle insulin.
• Human growth hormone benefits children with
hypopituitarism, a form of dwarfism.
• Tissue plasminogen activator (TPA) helps dissolve blood
clots and reduce the risk of future heart attacks.
• However, like many such drugs, it is expensive.
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• New pharmaceutical products are responsible for
novel ways of fighting diseases that do not respond
to traditional drug treatments.
• One approach is to use genetically engineered proteins
that either block or mimic surface receptors on cell
membranes.
• For example, one experimental drug mimics a receptor
protein that HIV bonds to when entering white blood
cells, but HIV binds to the drug instead and fails to
enter the blood cells.
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• Virtually the only way to fight viral diseases is by
vaccination.
• A vaccine is a harmless variant or derivative of a
pathogen that stimulates the immune system.
• Traditional vaccines are either particles of virulent
viruses that have been inactivated by chemical or
physical means or active virus particles of a
nonpathogenic strain.
• Both are similar enough to the active pathogen to
trigger an immune response.
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• Recombinant DNA techniques can generate large
amounts of a specific protein molecule normally
found on the pathogen’s surface.
• If this protein triggers an immune response against the
intact pathogen, then it can be used as a vaccine.
• Alternatively, genetic engineering can modify the
genome of the pathogen to attenuate it.
• These attenuated microbes are often more effective than
a protein vaccine because it usually triggers a greater
response by the immune system.
• Pathogens attenuated by gene-splicing techniques may
be safer than the natural mutants traditionally used.
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2. DNA technology offers forensic,
environmental, and agricultural
applications
• In violent crimes, blood, semen, or traces of other
tissues may be left at the scene or on the clothes or
other possessions of the victim or assailant.
• If enough tissue is available, forensic laboratories
can determine blood type or tissue type by using
antibodies for specific cell surface proteins.
• However, these tests require relatively large amounts of
fresh tissue.
• Also, this approach can only exclude a suspect.
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• DNA testing can identify the guilty individual with
a much higher degree of certainty, because the
DNA sequence of every person is unique (except
for identical twins).
• RFPL analysis by Southern blotting can detect
similarities and differences in DNA samples and
requires only tiny amount of blood or other tissue.
• Radioactive probes mark electrophoresis bands that
contain certain RFLP markers.
• Even as few as five markers from an individual can be
used to create a DNA fingerprint.
• The probability that two people (that are not identical
twins) have the same DNA fingerprint is very small.
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• DNA fingerprints can be used forensically to
presence evidence to juries in murder trials.
• This autoradiograph of RFLP bands of samples from a
murder victim, the defendant, and the defendant’s clothes
is consistent with the conclusion that the blood on the
clothes is from the victim, not the defendant.
Fig. 20.17
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• The forensic use of DNA fingerprinting extends
beyond violent crimes.
• For instance, DNA fingerprinting can be used to settle
conclusively a question of paternity.
• These techniques can also be used to identify the
remains of individuals killed in natural or man-made
disasters.
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• Variations in the lengths of satellite DNA are increasingly used as
markers in DNA fingerprinting.
• When pieces of DNA are centrifuged, a "satellite" band
develops that is separate from the rest of the DNA. This layer
is composed of simple sequence DNA.
• They have repeating units of only a few base pairs and are
highly variable from person to person.
• Individuals may vary in the numbers of repeats, simple
tandem repeats (STRs), at a locus.
• Restriction fragments with STRs vary in size among
individuals because of differences in STR lengths.
• PCR is often used to amplify selectively particular STRs or
other markers before electrophoresis, especially if the DNA
is poor or in minute quantities.
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• The DNA fingerprint of an individual would be truly
unique if it were feasible to perform restriction
fragment analysis on the entire genome.
• In practice, forensic DNA tests focus on only about five
tiny regions of the genome.
• The probability that two people will have identical DNA
fingerprints in these highly variable regions is typically
between one in 100,000 and one in a billion.
• The exact figure depends on the number of markers and
the frequency of those markers in the population.
• Despite problems that might arise from insufficient
statistical data, human error, or flawed evidence, DNA
fingerprinting is now accepted as compelling evidence.
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• Increasingly, genetic engineering is being applied
to environmental work.
• Scientists are engineering the metabolism of
microorganisms to help cope with some
environmental problems.
• For example genetically engineered microbes that can
extract heavy metals from their environments and
incorporate the metals into recoverable compounds may
become important both in mining materials and
cleaning up highly toxic mining wastes.
• In addition to the normal microbes that participate in
sewage treatment, new microbes that can degrade other
harmful compounds are being engineered.
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• For many years scientists have been using DNA
technology to improve agricultural productivity.
• DNA technology is now routinely used to make
vaccines and growth hormones for farm animals.
• Transgenic organisms with genes from another species
have been developed to exploit the attributes of the new
genes (for example, faster growth, larger muscles).
• Other transgenic organisms are
pharmaceutical “factories” - a
producer of large amounts of
an otherwise rare substance
for medical use.
Fig. 20.18
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• The human proteins produced by farm animals
may or may not be structurally identical to natural
human proteins.
• Therefore, they have to be tested very carefully to
ensure that they will not cause allergic reactions or
other adverse effects in patients receiving them.
• In addition, the health and welfare of transgenic farm
animals are important issues, as they often suffer from
lower fertility or increased susceptibility to disease.
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• To develop a transgenic organism, scientists remove
ova from a female and fertilize them in vitro.
• The desired gene from another organism are cloned and
then inserted into the nuclei of the eggs.
• Some cells will integrate the foreign DNA into their
genomes and are able to express its protein.
• The engineered eggs are then surgically implanted in a
surrogate mother.
• If development is successful, the results is a transgenic
animal, containing a genes from a “third” parent, even
from another species.
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• Agricultural scientists have engineered a number
of crop plants with genes for desirable traits.
• These includes delayed ripening and resistance to
spoilage and disease.
• Because a single transgenic plant cell can be grown in
culture to generate an adult plant, plants are easier to
engineer than most animals.
• The Ti plasmid, from the soil bacterium
Agrobacterium tumefaciens, is often used to
introduce new genes into plant cells.
• The Ti plasmid normally integrates a segment of its
DNA into its host plant and induces tumors.
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• Foreign genes can be inserted into the Ti plasmid
(a version that does not cause disease) using
recombinant DNA techniques.
• The recombinant plasmid can be put back into
Agrobacterium, which then infects plant cells, or
introduced directly into plant cells.
Fig. 20.19
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• The Ti plasmid can only be used as a vector to
transfer genes to dicots (plants with two seed
leaves).
• Monocots, including corn and wheat, cannot be infected
by Agrobacterium (or the Ti plasmid).
• Other techniques, including electroporation and DNA
guns, are used to introduce DNA into these plants.
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• Genetic engineering is quickly replacing traditional
plant-breeding programs.
• In the past few years, roughly half of the soybeans and
corn in America have been grown from genetically
modified seeds.
• These plants may receive genes for resistance to weedkilling herbicides or to infectious microbes and pest
insects.
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• Scientists are using gene transfer to improve the
nutritional value of crop plants.
• For example, a transgenic rice plant has been developed
that produces yellow grains containing beta-carotene.
• Humans use beta-carotene to make vitamin A.
• Currently, 70% of children
under the age of 5 in
Southeast Asia are deficient
in vitamin A, leading to
vision impairment and
increased disease rates.
Fig. 20.20
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• An important potential use of DNA technology
focuses on nitrogen fixation.
• Nitrogen fixation occurs when certain bacteria in the soil
or in plant roots convert atmospheric nitrogen to nitrogen
compounds that plants can use.
• Plants use these to build nitrogen-containing compounds,
such as amino acids.
• In areas with nitrogen-deficient soils, expensive
fertilizers must be added for crops to grow.
• Nitrogen fertilizers also contribute to water pollution.
• DNA technology offers ways to increase bacterial
nitrogen fixation and eventually, perhaps, to engineer
crop plants to fix nitrogen themselves.
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• DNA technology has led to new alliances between
the pharmaceutical industry and agriculture.
• Plants can be engineered to produce human proteins for
medical use and viral proteins for use as vaccines.
• Several such “pharm” products are in clinical trials,
including vaccines for hepatitis B and an antibody that
blocks the bacteria that cause tooth decay.
• The advantage of “pharm” plants is that large amounts
of these proteins might be made more economically by
plants than by cultured cells.
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3. DNA technology raises important safety
and ethical questions
• The power of DNA technology has led to worries
about potential dangers.
• For example, recombinant DNA technology may create
hazardous new pathogens.
• In response, scientists developed a set of guidelines
that have become formal government regulations in
the United States and some other countries.
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• Strict laboratory procedures are designed to protect
researchers from infection by engineered microbes
and to prevent their accidental release.
• Some strains of microorganisms used in
recombinant DNA experiments are genetically
crippled to ensure that they cannot survive outside
the laboratory.
• Finally, certain obviously dangerous experiments
have been banned.
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• Today, most public concern centers on genetically
modified (GM) organisms used in agriculture.
• “GM organisms” have acquired one or more genes
(perhaps from another species) by artificial means.
• Genetically modified animals are still not part of our
food supply, but GM crop plants are.
• In Europe, safety concerns have led to pending new
legislation regarding GM crops and bans on the import
of all GM foodstuffs.
• In the United States and other countries where the GM
revolution had proceeded more quietly, the labeling of
GM foods is now being debated.
• This is required by exporters in a Biosafety Protocol.
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• Advocates of a cautious approach fear that GM
crops might somehow be hazardous to human
health or cause ecological harm.
• In particular, transgenic plants may pass their new
genes to close relatives in nearby wild areas through
pollen transfer.
• Transference of genes for resistance to herbicides,
diseases, or insect pests may lead to the development of
wild “superweeds” that would be difficult to control.
• To date there is little good data either for or against
any special health or environmental risks posed by
genetically modified crops.
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• Today, governments and regulatory agencies are
grappling with how to facilitate the use of
biotechnology in agriculture, industry, and
medicine while ensuring that new products and
procedures are safe.
• In the United States, all projects are evaluated for
potential risks by various regulatory agencies, including
the Environmental Protection Agency, the National
Institutes of Health, and the Department of Agriculture.
• These agencies are under increasing pressures from
some consumer groups.
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• As with all new technologies, developments in
DNA technology have ethical overtones.
• Who should have the right to examine someone else’s
genes?
• How should that information be used?
• Should a person’s genome be a factor in suitability for a
job or eligibility for life insurance?
• The power of DNA technology and genetic
engineering demands that we proceed with
humility and caution.
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